Prerequisites for the pharmaceutical industry to develop and commercialise helminths and helminth-derived product therapy

During the past 10 years, immunologists, epidemiologists and parasitologists have made many new exciting discoveries in the field of helminth-mediated immune regulation. In addition, many animal experiments have shown that certain helminths or products derived from helminths can protect mice from developing allergic or autoimmune disease. Some clinical trials utilising Trichuris suis or Necator americanus for the treatment of allergic disorders and inflammatory bowel disease have been conducted. The outcomes of these trials suggest that they may be used to treat these disorders. However, to date no helminth therapy is routinely being applied to patients and no helminth-derived product therapy has been developed. In order to bring new drugs to the market and shoulder the enormous costs involved in developing such therapies, pharmaceutical companies need to be involved. However, currently the resources from the pharmaceutical industry devoted to this concept are relatively small and there are good reasons why the industry may have been reluctant to invest in developing these types of therapies. In this review article, the hurdles that must be overcome before the pharmaceutical industry might invest in these novel therapies are outlined.     

Ascaris lumbricoides and Trichuris trichiura infections associated with wastewater and human excreta use in agriculture in Vietnam

BackgroundWe assessed the risk of helminth infections in association with the use of wastewater and excreta in agriculture in Hanam province, northern Vietnam. In two cross-sectional surveys, we obtained samples from 1,425 individuals from 453 randomly selected households. Kato-Katz thick smear and formalin-ether concentration techniques were used for helminth diagnosis in two stool samples per person. Socio-demographic and water, sanitation and hygiene related characteristics, including exposure to human and animal excreta and household wastewater management, were assessed with a questionnaire.ResultsOverall 47% of study participants were infected with any helminth (Ascaris lumbricoides 24%, Trichuris trichiura 40% and hookworm 2%). Infections with intestinal protozoa were rare (i.e. Entamoeba histolytica 6%, Entamoeba coli 2%, Giardia lamblia 2%, Cryptosporidium parvum 5% and Cyclospora cayetanensis 1%). People having close contact with polluted Nhue River water had a higher risk of helminth infections (odds ratio [OR] = 1.5, 95% confidence interval [CI] 1.1–2.2) and A. lumbricoides (OR = 2.1, 95% CI 1.4–3.2), compared with those without contact. The use of human excreta for application in the field had an increased risk for a T. trichiura infection (OR = 1.5, 95% CI 1.0–2.3). In contrast, tap water use in households was a protective factor against any helminth infection (i.e. T. trichiura OR = 0.6, 95% CI 0.4–0.9). Prevalences increased with age and males had generally lower prevalences (OR = 0.8, 95% CI 0.6–1.0), participants performing agricultural (OR = 1.5, 95% CI 1.1–2.1) and having a low educational level (OR = 1.7, 95% CI 1.2–2.4) were significantly associated with helminth infections. None of the factors related to household's sanitary condition, type of latrine, household's SES, use of animal excreta, and personal hygiene practices were statistically significant associated with helminth infection.ConclusionsOur study suggests that in agricultural settings, direct contact with water from Nhue River and the use of human excreta as fertiliser in the fields are important risk factors for helminth infection. Daily use of clean water is likely to reduce the risk of worm infection. Deworming policies and national programs should give more attention to these agricultural at risk populations.     

Translatability of helminth therapy in inflammatory bowel diseases

Modern hygienic lifestyles are associated with the emergence of inflammatory bowel disease (IBD) which now afflicts millions of people in highly-developed countries. Meticulous hygiene interrupts conduits of transmission required for ubiquitous exposure to parasitic worms (helminths). We proposed that loss of exposure to helminths permits development of IBD. Early clinical trials suggested that exposure to helminths such as Trichuris suis or Necator americanus can improve IBD. Over the last several years, processes to “medicinalize” T. suis have been developed and use of this helminth is now being studied in large multi-center clinical trials. Concurrently, we and others have identified some of the immune regulatory mechanisms elicited by helminth exposure that suppress inappropriate intestinal inflammation. These efforts could soon result in new therapies for patients with IBD.     

Helminths as governors of immune-mediated inflammation

Immune-mediated diseases (e.g. inflammatory bowel disease, asthma, multiple sclerosis and autoimmune diabetes) are increasing in prevalence and emerge as populations adopt meticulously hygienic lifestyles. This change in lifestyles precludes exposure to helminths (parasitic worms). Loss of natural helminth exposure removes a previously universal Th2 and regulatory immune biasing imparted by these organisms. Helminths protect animals from developing immune-mediated diseases (colitis, reactive airway disease, encephalitis and diabetes). Clinical trials show that exposure to helminths can reduce disease activity in patients with ulcerative colitis or Crohn's disease. This paper summarises work by multiple groups demonstrating that colonization with helminths alters immune reactivity and protects against disease from dysregulated inflammation.     

Hypotensive effect of Oxacom® containing a dinitrosyl iron complex with glutathione: Animal studies and clinical trials on healthy volunteers

A comparative study of hypotensive effects of binuclear forms of dinitrosyl iron complexes (DNICs) with glutathione, S-nitrosoglutathione (GS-NO) and sodium nitrite (NaNO₂) on rats has been carried out. The latter appeared to be the least efficient, viz., mean arterial pressure (MAP) decreased by 10 and 30 mm Hg at 25 and 100 μmoles/kg of NaNO₂. In contrast, DNIC and GS-NO produced an appreciable hypotensive effect when used at much lower concentrations. GS-NO reduced MAP to the same extent, viz., to 90 mm Hg, on a hundredfold dose scale (from 0.4 up to 50 μmoles/kg) with subsequent restoration of MAP within the next 6–15 min. A similar effect was observed for DNIC except that the amplitude of the MAP drop was lower and the duration of hypotension was essentially greater. DNIC with glutathione were selected as a basic material for pilot-scale production of a hypotensive drug (commercial name Oxacom®). Preliminary pharmacological testing of Oxacom did not establish any adverse or deleterious side effects.Clinical trials of Oxacom® were performed on 14 healthy male volunteers in whom single intravenous infusion of the drug (5 mg/kg or 0.2 μmoles/kg of DNIC, respectively) evoked a characteristic response manifested as a 3–4 min drop by 24–27 mm Hg of both diastolic and systolic AP with its subsequent slow restoration within the next 8–10 h. The heart rate was quickly normalized after an initial increase. Cardiac output was unchanged despite reduced cardiac filling. A comprehensive analysis of clinical and biochemical data failed to establish any significant pathological changes in these parameters. The data obtained suggest that Oxacom® can be recommended for the second phase of clinical trials.     

Socio-medical studies of individuals self-treating with helminths provide insight into clinical trial design for assessing helminth therapy

The virtually complete loss of intestinal worms, known as helminths, from Western society has resulted in elimination of a range of helminth-induced morbidities. Unfortunately, that loss has also led to inflammation-associated deficiencies in immune function, ultimately contributing to widespread pandemics of allergies, autoimmunity, and neuropsychiatric disorders. Several socio-medical studies have examined the effects of intentional reworming, or self-treatment with helminths, on a variety of inflammation-related disorders. In this study, the latest results from ongoing socio-medical studies are described. The results point toward two important factors that appear to be overlooked in some if not most clinical trials. Specifically, (a) the method of preparation of the helminth can have a profound effect on its therapeutic efficacy, and (b) variation between individuals in the effective therapeutic dosage apparently covers a 10-fold range, regardless of the helminth used. These results highlight current limits in our understanding of the biology of both hosts and helminths, and suggest that information from self-treatment may be critical for clinical evaluation of the benefits and limits of helminth therapy.     

Helminthic therapy: improving mucosal barrier function

The epidemiology of autoimmune diseases and helminth infections led to suggestions that helminths could improve inflammatory conditions, which was then tested using animal models. This has translated to clinical investigations aimed at the safe and controlled reintroduction of helminthic exposure to patients suffering from autoimmune diseases (so-called 'helminthic therapy') in an effort to mitigate the inflammatory response. In this review, we summarize the results of recent clinical trials of helminthic therapy, with particular attention to mechanisms of action. Whereas previous reviews have emphasized immune regulatory mechanisms activated by helminths, we propose that enhancement of mucosal barrier function may have an equally important role in improving conditions of inflammatory bowel diseases.     

Cestode regulation of inflammation and inflammatory diseases

Helminth parasites are masters of immune regulation; a likely prerequisite for long-term survival by circumventing their hosts’ attempt to eradicate them. From a translational perspective, knowledge of immune events as a response to infection with a helminth parasite could be used to reduce the intensity of unwanted inflammatory reactions. Substantial data have accumulated showing that inflammatory reactions that promote a variety of auto-inflammatory diseases are dampened as a consequence of infection with helminth parasites, via either the mobilization of an anti-worm spectrum of immune events or by the direct effect of secretory/excretory bioactive immunomodulatory molecules released from the parasite. However, many issues are outstanding in the definition of the mechanism(s) by which infection with helminth parasites can affect the outcome, positively or negatively, of concomitant disease. We focus on a subgroup of this complex group of metazoan parasites, the cestodes, summarizing studies from rodent models that illustrate if, and by what mechanisms, infection with tapeworms ameliorate or exaggerate disease in their host. The ability of infection with cestodes, or other classes of helminth, to worsen a disease course or confer susceptibility to intracellular pathogens should be carefully considered in the context of ‘helminth therapy’. In addition, poorly characterised cestode extracts can regulate murine and human immunocyte function, yet the impact of these in the context of autoimmune or allergic diseases is poorly understood. Thus, studies with cestodes, as representative helminths, have helped cement the concept that infection with parasitic helminths can inhibit concomitant disease; however, issues relating to long-term effects, potential side-effects, mixed pathogen infections and purification of immunomodulatory molecules from the parasite remain as challenges that need to be addressed in order to achieve the use of helminths as anti-inflammatory agents for human diseases.     

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch,Perca fluviatilis

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated.Plasma cortisol and glucose levels increased markedly 1 h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1–72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses.Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.     

Fasciola hepatica: The therapeutic potential of a worm secretome

The success of helminth parasites is partly related to their ability to modulate host immune responses towards an anti-inflammatory/regulatory phenotype. This ability resides with the molecules contained in the secretome of various helminths that have been shown to interact with host immune cells and influence their function. Consequently, there exists a unique opportunity to exploit these molecules for the prophylactic and therapeutic treatment of human pro- and auto-inflammatory disorders (for example septic shock, transplant rejection and autoimmune disease). In this review, we describe the mechanisms used by the trematode parasite, Fasciola hepatica, to modulate the immune responses of its host and discuss the potent immune-modulatory effects of three individual molecules within the secretome; namely cathepsin L1, peroxiredoxin and helminth defence molecule. With a focus on the requirements from industry, we discuss the strategies by which these molecules may be clinically developed to control human immune responses in a way that is conducive to the prevention of immune-mediated diseases.     

The neonicotinoids thiacloprid,imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.)

A strong immune defense is vital for honey bee health and colony survival. This defense can be weakened by environmental factors that may render honey bees more vulnerable to parasites and pathogens. Honey bees are frequently exposed to neonicotinoid pesticides, which are being discussed as one of the stress factors that may lead to colony failure. We investigated the sublethal effects of the neonicotinoids thiacloprid, imidacloprid, and clothianidin on individual immunity, by studying three major aspects of immunocompetence in worker bees: total hemocyte number, encapsulation response, and antimicrobial activity of the hemolymph. In laboratory experiments, we found a strong impact of all three neonicotinoids. Thiacloprid (24 h oral exposure, 200 μg/l or 2000 μg/l) and imidacloprid (1 μg/l or 10 μg/l) reduced hemocyte density, encapsulation response, and antimicrobial activity even at field realistic concentrations. Clothianidin had an effect on these immune parameters only at higher than field realistic concentrations (50–200 μg/l). These results suggest that neonicotinoids affect the individual immunocompetence of honey bees, possibly leading to an impaired disease resistance capacity.     

TOPS-MODE model of multiplexing neuroprotective effects of drugs and experimental-theoretic study of new 1,3-rasagiline derivatives potentially useful in neurodegenerative diseases

The interest on computational techniques for the discovery of neuroprotective drugs has increased due to recent fail of important clinical trials. In fact, there is a huge amount of data accumulated in public databases like CHEMBL with respect to structurally heterogeneous series of drugs, multiple assays, drug targets, and model organisms. However, there are no reports of multi-target or multiplexing Quantitative Structure–Property Relationships (mt-QSAR/mx-QSAR) models of these multiplexing assay outcomes reported in CHEMBL for neurotoxicity/neuroprotective effects of drugs. Accordingly, in this paper we develop the first mx-QSAR model for multiplexing assays of neurotoxicity/neuroprotective effects of drugs. We used the method TOPS-MODE to calculate the structural parameters of drugs. The best model found correctly classified 4393 out of 4915 total cases in both training and validation. This is representative of overall train and validation Accuracy, Sensitivity, and Specificity values near to 90%, 98%, and 80%, respectively. This dataset includes multiplexing assay endpoints of 2217 compounds. Every one compound was assayed in at least one out of 338 assays, which involved 148 molecular or cellular targets and 35 standard type measures in 11 model organisms (including human). The second aim of this work is the exemplification of the use of the new mx-QSAR model with a practical case of study. To this end, we obtained again by organic synthesis and reported, by the first time, experimental assays of the new 1,3-rasagiline derivatives 3 different tests: assay (1) in absence of neurotoxic agents, (2) in the presence of glutamate, and (3) in the presence of H₂O₂. The higher neuroprotective effects found for each one of these assays were for the stereoisomers of compound 7: compound 7b with protection = 23.4% in assay (1) and protection = 15.2% in assay (2); and for compound 7a with protection = 46.2% in assay (3). Interestingly, almost all compounds show protection values >10% in assay (3) but not in the other 2 assays. After that, we used the mx-QSAR model to predict the more probable response of the new compounds in 559 unique pharmacological tests not carried out experimentally. The results obtained are very significant because they complement the pharmacological studies of these promising rasagiline derivatives. This work paves the way for further developments in the multi-target/multiplexing screening of large libraries of compounds potentially useful in the treatment of neurodegenerative diseases.     

Cerebral vasodilator properties of Danshen and Gegen: A study of their combined efficacy and mechanisms of actions

Danshen and Gegen are two commonly used Chinese herbal medicines for treatment of cardiovascular diseases. The aim of the present study was to elucidate the combination effects of these two herbs on cerebral vascular tone and their underlying mechanisms of actions. Basilar artery rings were obtained from rats and precontracted with U46619. Cumulative administrations of aqueous extracts of Danshen, Gegen, or the two herbs combined (DG; ratio 7:3) produced concentration-dependent relaxation of the artery rings. Statistical analysis on these findings produced a combination index (CI) of 1.041 at ED₅₀, which indicates the two herbs produced additive vasodilator effects when used as a combined decoction. Removal of the endothelium had no effect on the vasodilator properties of Danshen, Gegen, and DG. However, their maximum effects (Imax) were significantly blunted by a K_ATP channel inhibitor glibenclamide, a non-selective K⁺ channel inhibitor tetraethylammonium (TEA), and by a combination of K⁺ channel inhibitors (glibenclamide + TEA + iberiotoxin + 4-aminopyridine + barium chloride). In addition, Danshen, Gegen, and DG produced augmentation of K_ATP currents and inhibited Ca²⁺ influx in vascular smooth muscle cells isolated from rat basilar arteries. Furthermore, these agents inhibited CaCl₂-induced contraction in the artery rings. In conclusion, the present study showed that Danshen and Gegen produced additive vasodilator effects on rat cerebral basilar arteries. These effects were independent of endothelium-derived relaxant factors (EDRF), but required the opening of K_ATP channels and inhibition of Ca²⁺ influx in the vascular smooth muscle cells. It is suspected that the cerebral vasodilator effects of Danshen and Gegen produced either on their own or in combination, can help patients with obstructive cerebrovascular diseases.     

The upper and lower segments of subscapularis muscle have different roles in glenohumeral joint functioning

Subscapularis muscle is divided into two independent segments, upper and lower (USUB and LSUB), but the role of each segment in glenohumeral functioning is unclear. We compared the electromyographic (EMG) activity of USUB and LSUB during a variety of shoulder movements, with and without an external translation force. Intramuscular electrodes were inserted in USUB and LSUB segments of 20 adults without pathology and EMG activity was measured in stabilization trials (with and without an anterior or posterior directed force at the humerus and isometric rotations) and two shoulder positions (shoulder neutral, abduction). Maximal voluntary isometric contraction (MVIC) trials were performed in abduction, internal and external rotation of the shoulder. In MVIC trials, USUB showed higher activity during internal rotation (p = 0.03), whereas LSUB showed higher activity during external rotation (p < 0.01). In stabilization trials, the interaction effects were significant for muscle segment × condition (p < 0.01), and approached significance for muscle segment × position (p = 0.06). In the neutral position, the pattern of activity for LSUB was similar to USUB. In the abducted position the LSUB, unlike USUB, was more active during external rotation (p = 0.06) and also showed increased activity in response to the posterior directed force at the humerus (p = 0.04). Our results suggest that USUB primarily acts as an agonist for internal rotation. In contrast LSUB was particularly active in external rotation in the abducted position and demonstrated increased EMG activity in response to the posteriorly directed force at the humerus in that position, suggesting more of a role in glenohumeral stabilization.     

Long-term cyclosporine treatment in non-transplanted rats and metabolic risk factors of vascular diseases

Cyclosporine (CsA) is an immunosuppressive agent frequently used in the clinic for prevention of allograft rejection and for the treatment of autoimmune diseases. Despite its desired action on the immune system, CsA treatment may present serious adverse effects, which are masked by the concomitant use of other drugs. The search for effective immunosuppression protocols which does not affect the quality of life of patients is driving research to investigate the CsA involvement in vascular diseases, frequent in patients under immunosuppression. Thus, 45 non-transplanted Wistar rats were treated for 8 weeks with vehicle or 5 or 15 mg/kg CsA (n = 15/group) by gavage administration to evaluate the specific influence of cyclosporine on the levels of risk factors (metabolic and inflammatory) of vascular disease and its mechanism of action. Therefore, serum insulin levels, glucose tolerance test, serum lipids profile, total homocysteine and fibrinogen levels were assessed. The biochemical alterations reported here suggest the development of a framework straight to diabetes. Glucose homeostasis was affected as indicated by decreased insulin levels and altered glucose tolerance test in CsA 15 mg/kg group compared to other groups. Serum insulin and total homocysteine levels presented a significant negative correlation (R = ? 0.76, P < 0.0001). Fibrinogen and serum lipids profiles were significantly increased in CsA 15 mg/kg group compared to other groups and correlated positively with total homocysteine levels. Considering the well-established correlation among insulin resistance, lipid and total homocysteine levels, hypercoagulability and atherosclerosis, we can assume that this protocol of long-term CsA treatment in non-transplanted rats alter biochemical parameters related to cardiovascular and cerebrovascular risk, mainly in CsA 15 mg/kg group. Insulin and tHcy serum levels appear to be central in this process.     

The effect of ruminoreticulum bypass in yarded lambs on the efficacy of oxfendazole against resistant Trichostrongylus spp. helminths

An observational study was performed to investigate the effects of yarding and ruminoreticulum bypass on the efficacy of oxfendazole against resistant Trichostrongylus spp. helminths. Ruminoreticulum bypass was observed in 9-month old Perendale lambs using video-taped fluoroscopy and the efficacy of oxfendazole was estimated using the faecal egg count reduction test. Yarding and withholding feed for 24 h before drenching with oxfendazole significantly enhanced the faecal egg count reduction for resistant Trichostrongylus spp. parasites (p < 0.05), but ruminoreticulum bypass of the drench in the yarded animals had no negative effect on anthelmintic efficacy (p > 0.05). The effect of ruminoreticulum bypass on the efficacy of oxfendazole may depend on the resistant parasite species present. The positive effects of yarding sheep for 24 h before drenching, on the efficacy of oxfendazole against resistant Trichostrongylus spp., probably outweigh any negative effects of ruminoreticulum bypass. However further studies are required, with different resistant parasite genera present, before firm recommendations can be made.     

An in vivo microdialysis measurement of harpagoside in rat blood and bile for predicting hepatobiliary excretion and its interaction with cyclosporin A and verapamil

Harpagoside, a major bioactive iridoid glucoside in genus Scrophularia, has been widely used in clinical practice for the treatment of pain in the joints and lower back for its neuroprotective and anti-inflammation activities. To investigate the pharmacokinetics and hepatobiliary excretion, an in vivo microdialysis method coupled with high performance liquid chromatography was developed to monitor the concentration of harpagoside in blood and bile. The harpagoside bile-to-blood distribution ratio (AUC_bile/AUC_blood) up to 986.28 ± 78.46 significantly decreased to 6.41 ± 0.56 or 221.20 ± 18.92 after co-administration of cyclosporin A or verapamil. The results indicated that harpagoside went through concentrative elimination from the bile which was probably regulated by P-glucoprotein, providing possible clinical trials of co-administration of transporter inhibitors to decrease drug efflux, thus to enhance the curative effects.     

Effects of soybean oligosaccharides on intestinal microbial communities and immune modulation in mice

BackgroundSoybean oligosaccharides (SBOSs) are potential prebiotics that may be used to improve immune function. Here, we investigated the effects of intragastric administration of SBOSs in mice to determine the effects on autochthonous intestinal microbial communities and immunological parameters. Results E: After 22-day administration, 4.0 g kg body weight (BW)⁻¹ SBOSs significantly enhanced the proliferation of bifidobacteria and lactic acid bacteria (LAB) as compared to the control. This dose of SBOSs also significantly increased numbers of enterococci and decreased numbers of Clostridium perfringens. Treatment with 4.0 g kg BW⁻¹ SBOSs also significantly increased the percentage of T-lymphocytes and lymphocyte proliferation as compared to the control, suggesting that SBOSs promoted cellular immunity in mice. Additionally, 4.0 g kg BW⁻¹ SBOSs induced significant differences in hemolysin production, natural killer (NK) cell activity, phagocytic activity, cytokine production, and immunoglobulin levels compared to the control. Conclusion: Our data demonstrated that intragastric administration of SBOSs at a dose of 4.0 g kg BW⁻¹ improved the numbers of beneficial intestinal microbes and enhanced immunological function of mice. Therefore, these data supported that SBOSs may have applications as a prebiotic to improve immune responses in humans. Further studies are warranted.     

Effects of 2-deoxy-D-glucose on the immune system of seabream (Sparus aurata L.)

Stressful situations are a major problem in aquaculture because they affect the immune system. 2-Deoxy-d-glucose (2-DG) is a derivative of a glucose analogue that reduces the availability of energy, thereby inhibiting cell metabolism so that it is unable to enter the glycolysis pathway. In this paper, 2-DG has been administered in order to study if the immune function is compromised during metabolic stress. Blood glucose level was measured as an indicator of the inhibition of glycolysis, and the effects of intraperitoneal administration of 2-DG on the main parameters of the humoral (complement, IgM levels and peroxidase activity in blood plasma) and cellular (respiratory burst, intracellular peroxidase level and phagocytosis activity) immune parameters of gilthead seabream (Sparus aurata, L) were evaluated. Furthermore, the expression levels of immune-associated genes (CSF-1R, NCCRP-1, Hep, TCR-β, IgM_H, MHC-IIα, C3 and IL-1β) were analyzed by real-time PCR in head-kidney. A total of 5 intraperitoneal injections were performed at 48 h intervals. Three experimental groups were established: a control group injected with phosphate buffer saline, group 2-DG 500 and group 2-DG 750 injected with 500 mg kg^?1 and 750 mg kg^?1 2-DG, respectively (N = 15). After the third and fourth injection, some specimens of both DG-treated groups died. Following the first and third injection, the blood glucose levels of both 2-DG treated groups increased to a statistically significant extent with respect to the control group. While the humoral immune parameters were not significantly affected as a consequence of 2-DG administration, the cellular activities of leucocytes were. The injection of 500 mg kg^?1 2-DG provoked up- or down-regulation of the immune-relevant genes analyzed, while the injection of 750 mg kg^?1 always caused down-regulation of these genes. The results suggest that 2-DG provokes metabolic stress, which reduces the activities carried out by immune cells (leucocytes) and induces down-regulation of the immune-relevant genes analyzed when the energy available to the cell decreases.